ProjectSummary LargenumbersofgenevariantswereidentifiedfromgenomicsequencingofCongenitalHeartDisease patients, but lack of functional verification in heart development precludes assigning ?disease gene? status. Genetic control of heart development is conserved from Drosophila to humans, thus investigations in flies can illuminate gene functions in human heart development and disease. We developed a gene validation system in Drosophila to screen large numbers of genes for roles in heart development, and quantitative analysis tools to assess multiple phenotypic parameters. We also developed novel strategies to test patient-derived genetic mutations in flies for in vivo evidence linking specific gene variants to disease. We observed that many histone-modifying genes mutated in disease patientshaverolesinflyheartdevelopment.Weproposestudiesdesignedtoelucidateancestralrolesof histone-modifying genes in heart development and disease, and to generate personalized fly heart diseasemodelsforspecificgenevariants.Usingthehigh-throughputDrosophilagenevalidationscreen for essential roles in heart development, we will test candidate disease genes identified from publicly available datasets and collaborators? unpublished datasets. We will also screen Drosophila genes encodingenzymesforhistonemethylation/demethylationandacetylation/deacetylationforrolesinheart development. Histone modifying genes validated by screening will be phenotyped using multiple quantifiable morphological and functional readouts. We will identify histone modifications that are most important for heart development. Genes will be prioritized based on multiple criteria, and for highest priority genes we will examine the transcriptional profile of heart tissue from flies in which the gene of interestwassilencedincardiaccells.Wewillidentifyconservedtargetsofhistonemodificationeffectsby comparing our results to data from murine models and patient tissue samples. We will generate transgenicandknock-inflymodelstoprovideinvivofunctionalevidenceforinvolvementofhighpriority gene variants in congenital heart disease. In pReplacement, we will express wild type or mutant transgene versions of a given human disease gene in the fly heart while simultaneously silencing the endogenous fly homolog. We will also generate ?knock-in? Drosophila models using CRISPR/Cas9- mediated gene editing. In this pCRISPR approach, the endogenous Drosophila homolog is precisely modifiedtoencodeaproteinwithaminoacidchangesidenticaltothoseencodedinthepatient-derived mutantallele.WewillalsouseDrosophilatomodelpolygenicdiseasebasedonselectedpatients,each ofwhomcarriesmultiplegenevariantsthatincludeonemutanthistone-modifyinggene.